PROJECT SUMMARY/ABSTRACT Pseudomonas aeruginosa (PA) is a versatile opportunistic pathogen that is a leading cause of hospital- acquired infections in immunocompromised patients and in patients with Cystic Fibrosis. PA antibiotic resistance continues to explode, making development of new therapeutic approaches a critical need. This gram-negative bacterium encodes an unusually large number of so called two-component signal transduction systems, the major signaling machinery by which bacteria sense and respond to changes in their external environment, including 4 chemosensory-like systems. A unique feature of chemosensory systems is their ability to undergo sensory adaption, a short-term memory process by which the chemosensory system returns to its pre-stimulus level despite ongoing exposure to the input signal. In the E. coli Che system, adaptation involves the reversible methylation and demethylation of one or more glutamyl residues on the MCP. This is accomplished through the enzymatic activity of the constitutively active CheR methyltransferase and the regulated activity of the CheB methylesterase through what is essentially a delayed negative feedback circuit. Importantly, the CheR and CheB homologs are conserved in a diverse array of chemosensory-like systems that differ from the E. coli paradigm in their inputs and outputs. Therefore, much remains to be learned about the mechanistic consequences and physiologic roles of adaptation outside of chemotaxis, for example during biofilm formation. Our lab and others have described the Chp chemosensory system, one of 4 chemosensory systems encoded in PA. We have recently discovered that the Chp chemosensory system functions as a mechanochemical signaling (MCS) system that senses surface contact through retraction of the polarly localized type IV pilus (TFP) adhesin. Subsequent phosphorelay through the Chp MCS leads to two outputs: (i) regulation of a unique form of surface locomotion, type IV pili (TFP)-dependent twitching motility, and (ii) transcription of >200 genes involved in acute virulence, quorum sensing, and initiation of biofilm formation. Subsequent biofilm formation requires a cyclic-di-GMP-activated program. Even though the Chp system encodes a presumptive methyltransferase and methylesterase, little is known about how sensory adaptation might play a role in regulating its outputs. This is intriguing as the Chp system responds to surface contact and not to chemical gradients. We hypothesize that the Chp MCS system utilizes sensory adaption to finely tune second messenger levels upon surface contact to facilitate the transition from planktonic growth to biofilm formation. In this proposal we will test the hypotheses that (1) PilK and ChpB are polarly localized proteins that function to methylate and demethylate PilJ; (2) PilK/ChpB/PilJ- mediated sensory adaptation in the Chp MCS regulates the amplitude and kinetics of the surface- activated virulence program and/or the dynamics of twitching motility; and (3) The Chp MCS sensory adaptation is required for the transition from planktonic growth to biofilm formation.